Summary

We describe a novel murine progenitor cell population localised to a
previously uncharacterised region between sebaceous glands and the hair
follicle bulge, defined by its reactivity to the thymic epithelial progenitor
cell marker MTS24. MTS24 labels a membrane-bound antigen present during the
early stages of hair follicle development and in adult mice. MTS24
co-localises with expression of α6-integrin and keratin 14, indicating
that these cells include basal keratinocytes. This novel population does not
express the bulge-specific stem cell markers CD34 or keratin 15, and is
infrequently BrdU label retaining. MTS24-positive and -negative keratinocyte
populations were isolated by flow cytometry and assessed for colony-forming
efficiency. MTS24-positive keratinocytes exhibited a two-fold increase in
colony formation and colony size compared to MTS24-negative basal
keratinocytes. In addition, both the MTS24-positive and CD34-positive
subpopulations were capable of producing secondary colonies after serial
passage of individual cell clones. Finally, gene expression profiles of MTS24
and CD34 subpopulations were compared. These results showed that the overall
gene expression profile of MTS24-positive cells resembles the pattern
previously reported in bulge stem cells. Taken together, these data suggest
that the cell-surface marker MTS24 identifies a new reservoir of hair follicle
keratinocytes with a proliferative capacity and gene expression profile
suggestive of progenitor or stem cells.

INTRODUCTION

The epidermis is a complex tissue consisting of a stratified squamous
epithelium (interfollicular epidermis), hair follicles and glandular
structures that function together as an organism's main barrier against the
external environment. Because of continual physical, chemical and biological
damage from the environment, the epidermis undergoes regular self-renewal.
Epidermal stem cells, which form the basis of this system, reside in several
locations including the interfollicular epidermis, sebaceous glands, and hair
follicles (Niemann and Watt,
2002; Fuchs et al.,
2004; Moore and Lemischka,
2006). Hair follicles are multilayered epidermal appendages of
several concentric layers that undergo a carefully regulated growth cycle
divided into phases of active growth (anagen), regression (catagen) and rest
(telogen) (Hardy, 1992).

Currently, there are several methods to experimentally distinguish
epidermal stem cells from the cycling transit amplifying cells. One approach
is to pulse-label neonatal mice repeatedly with injections of
[3H]thymidine or 5-bromo-2′-deoxyuridine (BrdU). Using this
method, all the actively dividing cells in the epidermis are labelled at a
time when the skin is hyperproliferative. This pulse is followed by a long
chase period (4-10 weeks) during which the [3H]thymidine- or
BrdU-label is lost through proliferation-associated dilution. In contrast,
infrequently dividing stem cells retain the label and are therefore called
label-retaining cells (LRC) (Bickenbach,
1981; Cotsarelis et al.,
1990; Bickenbach and Chism,
1998; Lavker and Sun,
2000).

A second approach to distinguish epidermal stem cells from transit
amplifying cells in humans involves analysis of the proliferative potential of
single cultured cells. Analysis of the resulting epidermal clones led to
classification of keratinocytes into stem-like, highly proliferative
holoclones and more abortive mero- and paraclone colonies
(Barrandon and Green, 1987).
Several studies have shown that LRC isolated from skin of adult mice
(Morris and Potten, 1994) or
rats (Pavlovitch et al., 1991;
Kobayashi et al., 1993) are
also clonogenic in culture. These follicular keratinocytes were highly
proliferative, particularly in the rat where the hair follicle bulge region
contains predominantly (95%) clonogenic keratinocytes
(Kobayashi et al., 1993;
Oshima et al., 2001). The
multipotentiality of individual mouse pelage or rat vibrissal bulge stem cells
was demonstrated by mouse skin transplantation, where clonally derived cells
were able to give rise to new hair follicles
(Blanpain et al., 2004) or
contribute to endogenous developing follicles
(Claudinot et al., 2005).

Epidermal stem cells have also been distinguished from transit amplifying
cells by their unique cell phenotype. Initially, human epidermal stem cells
and transit amplifying cells were distinguished by differential expression of
integrins and keratins. Human epidermal stem cells revealed a higher
expression of β1, α2, α3 and α6-integrin compared to
transit amplifying cells (Jones et al.,
1995; Tani et al.,
2000; Akiyama et al.,
2000; Braun et al.,
2003). Murine epidermal stem cells have been characterised by a
strong expression of keratin 15 (K15) (Liu
et al., 2003; Morris et al.,
2004), although this marker may not be exclusive to stem cells in
all situations (Amoh et al.,
2005). Expression of α6-integrin (in combination with a low
expression of the transferrin receptor CD71) and K19 have been correlated with
[3H]thymidine-label-retaining-cells, indicating that these markers
can identify murine epidermal stem cells
(Michel et al., 1996;
Tani et al., 2000). Another
approach has been to examine candidate cell-surface markers that identify stem
cells in other tissues. The cell-surface glycoprotein CD34 is expressed on
early hematopoietic progenitor cells, and its use in the purification of stem
cells for bone marrow transplants has been well established
(Brown et al., 1991;
Krause et al., 1994). More
recently, CD34 was shown to be expressed in the hair follicle bulge of murine
skin and CD34-positive cells, purified by fluorescence-activated cell sorting
(FACS), were shown to have clonogenic potential in vitro
(Trempus et al., 2003).

Recent evidence suggests that epidermal keratinocytes are capable of
recruiting hematopoietic precursors and supporting development of a thymic
microenvironment (Clark et al.,
2005). These data suggest potential functional and phenotypic
links may exist between progenitor cells in epidermal and thymic epithelia.
Several years ago, a specific monoclonal antibody marker was described for
epithelial progenitor cells in the mouse thymus. This marker, MTS24,
identified a glycoprotein with a peptide backbone of ∼80 kD, which was
expressed on a rare subset of epithelial cells in the adult thymus
(Gill et al., 2002;
Bennett et al., 2002). During
the early embryonic development of the thymus, a large proportion of thymic
epithelial cells are reactive for MTS24. Transplantation of purified fetal
MTS24-positive thymic epithelial cells under the kidney capsule generated a
normal microenvironment, indicating that MTS24-positive thymic epithelial
cells comprise a population of precursor cells capable of recruiting
hematopoietic precursors and giving rise to a fully functional thymic
epithelium.

Here, we report that the cell-surface marker MTS24 identifies a previously
uncharacterised population of hair follicle keratinocytes located between the
bulge and the sebaceous glands. MTS24 reactivity is first detected in the
early stages of hair follicle development, and is increased during hair
growth. MTS24-positive keratinocytes are distinct from the epidermal stem
cells located in the bulge, but exhibit increased colony-forming efficiency in
culture versus normal basal keratinocytes. Furthermore, the gene expression
profile of MTS24-positive keratinocytes resembles the pattern previously
reported for epidermal bulge stem cells. Our results suggest that the
MTS24-positive keratinocytes represent an important new committed progenitor
or stem cell compartment within the hair follicle.

MATERIALS AND METHODS

Experimental mice

To assess and compare MTS24 in different mouse strains we obtained mice
with normal hair development (C57Bl/6 mice and Balb/c) and mice with abnormal
hair development (nude and SKH-1 hairless mice) at 6-8 weeks of age from
Charles River (Maastricht, The Netherlands). The SKH-1 mice are an
uncharacterised hairless strain of mice that go through one hair cycle, after
which they lose their fur and become hairless.

Mice were held in the animal facility of the Leiden University Medical
Centre under a 12-hour light-dark cycle at 23°C/60° humidity and given
food and water ad libitum in accordance with the university's ethical
committee guidelines on animal care. At Cancer Research UK all mouse husbandry
and experimental procedures were conducted in compliance with the CR-UK animal
ethics committee. The K14ΔNβ-cateninER transgenic mice were
generated as described previously (Lo
Celso et al., 2004). The K14ΔNβ-cateninER transgene was
activated by topical application of 4-hydroxytamoxifen (4OHT; Sigma) to a
clipped area of dorsal skin (1 mg per mouse; 3 treatments/week).

BrdU labelling

To generate label-retaining cells (LRC), we used the protocol as described
by Bickenbach and colleagues (Bickenbach et
al., 1986; Bickenbach and
Chism, 1998). Ten-day-old mice were injected with 50 mg/(kg body
weight) 5-bromo-2′-deoxyuridine (BrdU; 20 μl of 12.5 mg/ml BrdU)
every 12 hours for a total of four injections, to label mitotic cells.

Tissue preparation

Mice were killed with CO2. Dorsal skin and tail skin were
embedded in tissue-tek O.C.T. compound (Sakura Finetek Europe). Frozen
sections (5-7 μm) were fixed for 30 minutes in formaldehyde (1% in PBS).
Wholemounts from tail skin were prepared as described by Braun and colleagues
(Braun et al., 2003). Epidermal
wholemounts were fixed for a minimum of 10 minutes to 2 hours in formaldehyde
(1% in PBS).

Immuno-electronmicroscopy

For immuno-electronmicroscopy, dorsal skin obtained from a 2-day-old SKH-1
mouse was fixed in 2% paraformaldehyde in 0.1 M Sörensen phosphate buffer
(pH 7.2) for 2 hours at RT. Upon fixation, skin was cut into pieces of
1×1×1 mm3. Skin was cryoprotected in 2.3 M sucrose for
30 minutes and snap frozen in liquid nitrogen until further use. For
immuno-electronmicroscopy, ultrathin sections (45 nm, Leica ultracut UCT) were
incubated with MTS24 (1:50), followed by biotin-conjugated rabbit anti-rat
(1:100). To visualise MTS24 reactivity, 15-nm protein A gold (own fabricate)
was used (1:200). Between incubation steps, sections were washed with
PBS/glycin. After incubation, the sections were embedded in methylcellulose
and stained with uranylacetate. MTS24 reactivity was viewed with a Philips 410
electron microscope (Philips, Eindhoven, the Netherlands).

FACS and clonogenicity assay

Keratinocytes were isolated and cultured from dorsal skin of adult C57Bl/6
mice essentially as reported previously by Romero et al.
(Romero et al., 1999),
incorporating the modifications described by Silva-Vargas et al.
(Silva-Vargas et al., 2005).
To dual-label keratinocytes for α6-integrin/MTS24 orα
6-integrin/CD34, cell suspensions were incubated with either MTS24
(diluted 1:50 or 1:100) or biotinylated CD34 antibody for 20 minutes at
4°C and then washed with PBS. Cells were subsequently incubated with
RPE-conjugated donkey anti-rat IgG (to detect MTS24) or
streptavadin-conjugated RPE (to detect CD34). Cells were then washed and
blocked for 10 minutes in normal mouse serum (1:100) followed by incubation
with FITC-conjugated rat anti-human α6-integrin
(Trempus et al., 2003). Theα
6 antibody was used to select for basal keratinocytes, thus eliminating
suprabasal (differentiating) keratinocytes and non-keratinocytes from the
population collected. Cell viability was assessed by 7AAD (BD Biosciences)
staining. Dead cells and cells with high forward and side scatter were gated
out. Cells were sorted into supplemented, calcium-free FAD media containing
10% foetal bovine serum (Invitrogen) using a FACSVantage machine (Becton
Dickinson). Sorted populations were gated as follows: α6-integrin
single+, α6+/MTS24+, or α6+/CD34+ dual positive. Sorting gates
were drawn based upon staining intensity of single colour controls, and were
excluded in all regions from overlapping with negative and/or isotype
controls. One thousand keratinocytes were plated per 35 mm dish, and cultures
were maintained for 14 days. Cultures were fixed with 4% formal saline and
stained with 1% Rhodamine B. The area of the colonies was determined by using
EclipseNet Software (Nikon). Colony-forming efficiency was defined as the
percentage of cells forming a colony of three or more cells from the total
number of plated cells. For serial passaging experiments, 1000 sorted cells
were grown on 10 cm dishes for 10-14 days until colonies were visible. Plates
were washed with versene to remove feeders, and 5-10 randomly selected
colonies were individually trypsinised using cloning cylinders. Single
colonies were then transferred to secondary 35 mm dishes, and grown for an
additional 10-14 days, after which time the average number and surface area of
colonies was assessed. Two-tailed unpaired t tests were performed
with significance recognised with P<0.05 (GraphPad Software, San
Diego).

To perform FACS analysis for MTS24 and CD34, cells were labelled with the
primary antibody for MTS24 for 20 minutes at 4°C, washed twice in PBS and
incubated in AlexaFluor 488 goat anti-rat IgG. Cells were then washed and
blocked for 10 minutes in normal mouse serum (1:100) prior to incubation with
a biotinylated CD34 antibody followed by further washing and incubation with
streptavidin conjugated RPE.

RNA isolation and quantitative real-time PCR

We performed quantitative real-time PCR (Q-PCR) to determine the expression
of a selection of genes that were expected to be up- or downregulated in hair
follicle stem cells compared to non-stem cells
(Tumbar et al., 2004;
Morris et al., 2004;
Claudinot et al., 2005). Using
FACS (method described in previous section), we isolated α6-integrin
single+, α6+/MTS24+ and α6+/CD34+ keratinocytes obtained from skin
of C57Bl/6 mice (>100,000 cells per population). Total RNA was isolated
from the sorted cells (average yield was 110 ng total RNA/100,000 cells) with
the RNeasy Mini Kit (Qiagen) and mRNA amplification was performed with the
MessageAmp II aRNA Amplification Kit (Ambion) using T7-oligo-(dT) primers
according to the manufacturer's protocol. cDNA was synthesized from amplified
RNA with iScript Select cDNA synthesis kit (Bio-Rad) using random priming.
Q-PCR assays were performed on a MyIQ single colour real-time PCR (Bio-Rad)
using SYBR Green Supermix (Bio-Rad). PCR reaction was carried out according to
the following protocol: initial denaturation at 95°C (3 minutes) followed
by 40 cycles of 95°C (15 seconds) and 58°C (20 seconds). Primer
sequences can be provided on request. A melting curve was generated for each
product to ensure the specificity of the PCR product. Threshold cycles (Ct
values) were calculated using the MyIQ software (Bio-Rad). The reference gene
beta-actin was used to normalise the Ct values of the genes of interest
(ΔCt). Relative alterations (fold change) in mRNA expression levels inα
6+/MTS24+ and α6+/CD34+ keratinocytes were calculated according
to the algorithms
2-(ΔCt)α6+MTS24+/2-(ΔCt)α6+MTS24-
and
2-(ΔCt)α6+CD34+/2-(ΔCt)α6+CD34-
respectively. FACS isolation was performed in duplicate, and each Q-PCR
reaction was performed in triplicate.

RESULTS

Immunofluorescent staining of tissue sections
(Fig. 1A,B; red) or whole
mounts (Fig. 1F,G; green) of
tail skin from adult C57Bl/6 mouse showed bright staining of MTS24 in the hair
follicle. MTS24 was predominantly found in a previously uncharacterised region
of the hair follicle between the bulge and the sebaceous glands. The intensity
of this staining decreased towards the lower part and upper part of the hair
follicle, although labelling was occasionally seen in the infundibulum (the
upper part of the hair follicle), as well as in the cells at the perimeter of
the sebaceous gland (data not shown). A similar pattern of MTS24 labelling was
found in dorsal skin from Balb/c (Fig.
1C-E), SKH-1 hairless mice
(Fig. 1H-J) and nude mice (data
not shown). Higher magnification revealed that MTS24 was primarily located on
the membrane of hair follicle cells (Fig.
1E). However, along the inner hair shaft MTS24 staining was not
membrane-bound but had a more smear-like appearance
(Fig. 1H, asterisk). The hair
follicle bulb did not show any MTS24 labelling
(Fig. 1F). Negative controls
did not show non-specific staining of MTS24 (data not shown).

The staining pattern of MTS24 in murine skin of 2-day-old SKH-1 mice
(Fig. 1J) was further
characterised using immuno-electronmicroscopy
(Fig. 1K-N). A cross-sectional
overview of the murine hair follicle (Fig.
1M) showed that MTS24 reactivity was found in both the outer root
sheath (ORS; see also Fig. 1K)
and the inner root sheath (IRS; see also
Fig. 1L,N). Within the ORS,
metabolising cells were found, characterised by the presence of
heterochromatin (Fig. 1K,M;
asterisks). These cells showed membrane-bound staining for MTS24, as indicated
by the gold particles associated with their cell membrane
(Fig. 1K, arrowheads). Within
the IRS, apoptotic cells were found (Fig.
1L,M; crosshatch). These apoptotic cells also showed
membrane-bound MTS24 labelling (Fig.
1L). More centrally within the IRS, many tightly packed membranes
of dead cells were found, which the MTS24 antibody also labelled
(Fig. 1N). This observation
correlates with the smearing pattern of MTS24 as shown in
Fig. 1H. Taken together, these
data indicate that MTS24 labels a membrane-bound antigen that is localised in
a previously uncharacterised region of the murine hair follicle adjacent to
the bulge.

MTS24 and CD34 localisation during hair follicle development

To investigate the relationship between MTS24 expression reactivity and
murine hair follicle development, we analysed MTS24 labelling in murine dorsal
skin at different stages of hair follicle development. The timepoints ranged
from embryonal day 17 (E17), at which the largest group of hair follicles
(non-tylotrich follicles) start to develop
(Schmidt-Ullrich and Paus,
2005), to postnatal day 8, by which time all hair follicles are
fully developed. Recently, expression of the cell-surface glycoprotein CD34
has been reported in keratinocytes localised in the bulge region of the adult
hair follicle (Trempus et al.,
2003). To permit comparison of CD34 and MTS24 reactivity during
both normal and aberrant hair follicle development, we labelled frozen
sections of dorsal skin from Balb/c or SKH-1 mice with antibodies to MTS24 or
CD34, and co-stained with keratin 17 (K17), a marker exclusively expressed in
hair follicles of normal skin (Panteleyev
et al., 1997). We first observed weak co-localisation of MTS24 and
K17 in the developing hair follicle at E17 (data not shown). At E20.5 MTS24
staining was intense and showed co-localisation with K17 expression in the
developing hair follicle (Fig.
2A, upper panel). As hair follicle development progressed further,
both MTS24 and K17 labelling increased in intensity. At day 2 after birth,
MTS24 was found in the entire upper hair follicle as well as in the
interfollicular epidermis, both in the SKH-1 mouse
(Fig. 1J) and in Balb/c mouse
dorsal skin (Fig. 2A, second
panel, arrowhead). In all cases, the interfollicular labelling was
specifically localised to areas where new hair follicles were developing. From
7-8 days after birth, MTS24 labelling became completely restricted to the hair
follicle (Fig. 2A, third
panel).

In contrast to MTS24, which was first detectable at E17 and was clearly
visible at E20.5 during hair follicle development
(Fig. 2A), hair follicles in
neonatal Balb/c mice up to 4 days of age
(Fig. 2B) failed to show
labelling for CD34. However, at 6 days after birth CD34 labelling was observed
in the bulge region of the hair follicle
(Fig. 2C). In contrast to
MTS24, in adult SKH-1 hairless mice no labelling for CD34 was ever observed in
the hair follicle bulge (data not shown). These data indicate that MTS24 is
present at an earlier stage than CD34 expression in hair follicle
development.

MTS24 and CD34 reactivity during hair follicle development.
(A) Staining of MTS24-Cy3 (red) and keratin 17-FITC (green) in skin
obtained from Balb/c mice at E20.5 during embryonic development and at day 2
and day 8 after birth. Keratin 17 was selectively expressed within the
developing hair follicles. Note the interfollicular staining of MTS24 and its
co-localisation with keratin 17 expression in 2-day-old Balb/c mice
(arrowhead). (B,C) Expression of CD34-Cy3 (red), keratin 17-FITC
(green) in dorsal skin from Balb/c mice at day 4 (B) and day 6 (C). Note that
CD34 was expressed from day 6 after birth but not at day 4 after birth. Red
staining of stratum corneum is caused by autofluorescence. (D,E)
Frozen sections of dorsal epidermis from K14ΔNβ-cateninER
transgenic mice treated with 4OHT for 21 days were immunolabelled for MTS24
(green; D,E) and keratin 14 (red; D) or keratin 17 (red; E). MTS24-positive
regions of ectopic follicles are demarcated with brackets (D,E). Nuclei were
counterstained (blue) with DAPI (A-C) or ToPro3 (E). HF, hair follicle; IFE,
interfollicular epidermis; BG, bulge. Scale bars: 25 μm (A, upper and
second panel); 50 μm (A, third panel; B-E).

It has been reported that activation of β-catenin in the epidermis of
K14ΔNβ-cateninER adult transgenic mice by topical application of
4-hydroxytamoxifen (4OHT) results in the formation of ectopic hair follicles
from sebaceous glands, interfollicular epidermis and pre-existing hair
follicles (Van Mater et al.,
2003; Lo Celso et al.,
2004; Silva-Vargas et al.,
2005). This expansion in the number of hair follicles is
associated with a dramatic increase in the percentage of CD34-reactive,
follicular stem-like cells in the skin
(Silva-Vargas et al., 2005).
To assess whether MTS24 reactivity is similarly increased during de novo hair
follicle formation, we stained frozen sections of dorsal tissue collected from
K14ΔNβ-cateninER transgenic mice following thrice-weekly treatment
with 4OHT for 21 days. MTS24 reactivity was found in ectopic follicles formed
from both interfollicular epidermis and pre-existing follicles
(Fig. 2D). During the early
stages of follicle neogenesis, MTS24 was present throughout the developing
follicle (data not shown), reminiscent of the staining pattern observed during
embryonic development (Fig.
2A). As the follicles developed further, MTS24 began to be
restricted to a mid-region of the K17-positive follicle
(Fig. 2E). In addition, we
assessed the localisation of MTS24 labelling in normally cycling hairs (see
Fig. S1 in the supplementary material), demonstrating that expression is
increased during anagen. In summary, these findings indicate that MTS24
reactivity is increased during de novo hair follicle formation and during the
growth (anagen) phase of the hair cycle.

MTS24 localisation with described stem cell markers

We used several described markers of the epidermal stem cell compartment to
examine their co-localisation with MTS24. K14 is expressed in all
keratinocytes in the basal layer of interfollicular epidermis as well as in
the outer root sheath of the hair follicle
(Fig. 3A). Immunolabelling of
frozen sections of dorsal epidermis showed that K14 and MTS24 co-localise
within the hair follicle, demonstrating that MTS24-positive cells are
keratinocytes (Fig. 3A-C).
Label-retaining cells tend to be clustered in the hair follicle bulge
(Cotsarelis et al., 1990), a
region that also expresses high levels of the markers CD34, keratin 15, andα
6-integrin (Lyle et al.,
1998; Trempus et al.,
2003; Morris et al.,
2004). In wild-type dorsal mouse skin, expression of CD34
(Fig. 3D, red, arrowhead) in
the hair follicle was adjacent to, but did not co-localise with MTS24
(Fig. 3E, green, asterisk).
CD34 expression was found directly beneath the MTS24 region
(Fig. 3F, red versus green). In
whole mounts of tail epidermis, labelling for K15
(Fig. 3G, green) and MTS24
(Fig. 3H, red) did not
co-localise (Fig. 3I). A
negative control for keratin 15 (incubation without the primary antibody)
showed that the intense green staining within the sebaceous gland was
background staining due to use of an anti-mouse secondary antibody
(Fig. 3J). α6-Integrin
expression was very bright throughout the entire hair follicle, including the
bulge and the region where MTS24 was detected
(Fig. 3K, arrowhead).

To investigate whether MTS24-positive keratinocytes are rarely dividing
cells, we injected SKH-1 and CBAxC57Bl/6 10-day-old mice repeatedly with BrdU
to generate label-retaining cells (LRC). In SKH-1 mice MTS24-positive cells
were BrdU labelled at one day post-injection
(Fig. 3L). After a chase period
of 6 weeks, LRC were still occasionally found within the population of
MTS24-positive cells, although the majority of the cells have depleted their
label (Fig. 3M). In CBAxC57Bl/6
mice, after a 10 week chase, the region of the hair follicle that was MTS24
reactive contained some BrdU-positive cells, but most of the LRC were
clustered in the bulge region of the follicle, beneath the MTS24-positive
region of the follicle (Fig.
3N,O).

Taken together, these findings indicate that MTS24 labelling co-localised
with expression of the basal keratinocytes markers α6-integrin and
keratin 14 but not with the bulge-specific markers keratin 15 and CD34. BrdU
label-retaining cells occasionally were found within the MTS24-positive cell
population.

MTS24-positive keratinocytes form large colonies with high efficiency
in culture

FACS analysis was performed to compare immunolabelling of keratinocytes
incubated with isotype-specific control
(Fig. 4A) or MTS24
(Fig. 4B) antibodies. In this
representative experiment (Fig.
4B), 4.1% of the keratinocytes were gated as MTS24-positive;
typically in our experiments the MTS24-positive subpopulation of keratinocytes
ranged from 4% to 8%. We used FACS enrichment to determine whether
MTS24-positive keratinocytes possess a high in vitro proliferative potential,
a well-established characteristic of epithelial stem cells
(Kobayashi et al., 1993). Data
are shown from a representative experiment containing at least four replicates
for each sort condition indicated; this experiment was repeated three times
with similar results (Fig.
4C-F). Three groups of sorted keratinocytes were collected and
seeded at clonal density: the unfractionated `all sorted' population of
undifferentiated cells with low forward and side scatter, α6+/MTS24+
cells (6.2% of undifferentiated cells) and α6+/MTS24-cells (48.1% of
undifferentiated cells) (Fig.
4C). After 14 days in culture, all three populations formed
colonies, however the α6+/MTS24+ keratinocytes gave rise to colonies
with the greatest efficiency (Fig.
4D,E). In addition, there was enrichment for larger colonies from
the α6+/MTS24+ population (Fig.
4D,E). Statistical analysis demonstrated significant differences
in colony-forming efficiency (P<0.0003) and the average area of
the colonies (P<0.0003) between the α6+/MTS24+ andα
6+/MTS24-fractions (Fig.
4E, asterisks). We quantified the percentage of the total number
of colonies based upon the size of the colonies
(Fig. 4F). The results showed
that α6+/MTS24+ keratinocytes form abortive colonies (colony area <1
mm2) significantly less frequently thanα
6+/MTS24-keratinocytes (Fig.
4F; P<0.0001). In contrast, the α6+/MTS24+
fraction produced significantly more large colonies thanα
6+/MTS24-keratinocytes (colony area 3-4 mm2,
P<0.0008; colony area >4 mm2, P<0.02)
(Fig. 4F). These results
indicate that MTS24+ basal keratinocytes possess a higher degree of
proliferative potential when compared to normal basal keratinocytes.

A population of clonogenic CD34-positive basal keratinocytes has been
reported in the hair follicle bulge
(Trempus et al., 2003). To
assess whether CD34+ keratinocytes represent a unique population from MTS24+
cells, keratinocytes isolated from C57Bl/6 mouse dorsal skin were labelled
with antibodies to CD34 and MTS24. FACS analysis identified distinct
CD34+/MTS24-(0.67%) and CD34-/MTS24+ (4.4%) populations of cells
(Fig. 5A). We were unable to
detect any CD34+/MTS24+ dual-positive keratinocytes. To directly compare the
colony-forming potential of CD34+ versus MTS24+ basal keratinocytes, cells
isolated from dorsal skin of adult C57Bl/6 mice were immunolabelled with
antibodies against MTS24/α6-integrin or CD34/α6-integrin. FACS
sorting and culture conditions were performed as described in
Fig. 4. Data shown are
representative of three separate experiments with six replicates for each sort
condition indicated (Fig.
5B-E). After 14 days in culture, sorted α6+/CD34+
keratinocytes gave rise to the largest colonies which were composed of mainly
small, apparently undifferentiated keratinocytes
(Fig. 5B). The α6+/MTS24+
colonies were intermediate in terms of colony size and relative fraction of
small keratinocytes, while the α6-single positive fractions
predominantly generated small colonies composed of mostly large,
differentiated keratinocytes (Fig.
5B). Both the α6+/CD34+ and α6+/MTS24+ subpopulations
had increased relative colony-forming efficiency compared with the
unfractionated `all sorted' population
(Fig. 5C), although theα
6+/CD34+ sorted keratinocytes were more efficient at primary colony
formation (Fig. 5C).

CD34 and MTS24 identify distinct subpopulations of basal keratinocytes
with high in vitro replicative capacity. (A,B) Keratinocytes
harvested from dorsal epidermis of adult C57Bl/6 mice were sorted under
sterile conditions. (A) Flow cytometric analysis shows that CD34+ basal
keratinocytes are members of a different population of cells than the MTS24+
keratinocytes. Sorted α6 single+, α6+/MTS24+, α6+/CD34+ or
the unseparated mixture (all sorted) keratinocytes colonies were grown for 14
days to visualise the colonies (B) and compare relative colony-forming
efficiency (C). (D) Individual colonies from α6+/MTS24+
and α6+/CD34+ keratinocytes were passaged and replated at clonal density
for an additional 14 days. (E) A graphical comparison of the size of
colonies derived following passage. Bars represent the mean of at least four
replicate culture wells±s.e.m. Data are shown from representative
experiments that were repeated with similar results.

We next wished to determine whether isolated, sorted, and cultured
individual colonies were capable of giving rise to secondary colony cultures
with high efficiency. Individual colonies from α6+/MTS24+ andα
6+/CD34+ sorted keratinocytes were cultured for 10 days, isolated by
ring cloning, and re-plated on secondary dishes for an additional 14 days.
Both progenitor cell subpopulations efficiently generated secondary colonies
following serial passage (Fig.
5D), and average colony size was not significantly different
between bulge-derived CD34+ stem cells and MTS24+ cells
(Fig. 5E).

Gene expression profiling of MTS24 versus CD34 basal
keratinocytes

Microarray studies have revealed that hair follicle stem cells exhibit a
specific gene expression profile compared to non-bulge basal keratinocytes
(Morris et al., 2004;
Tumbar et al., 2004;
Claudinot et al., 2005). Based
on these data, we selected 13 genes that were described to be up- or
downregulated in hair follicle stem cells compared to non-stem cells. Using
Q-PCR we studied the expression profile of these selected genes in FACS-sortedα
6+/MTS24+ and α6+/CD34+ keratinocytes compared toα
6+/MTS24- and α6+/CD34-keratinocytes. Average data are shown from
two independent FACS sorting experiments and Q-PCR was performed in triplicate
per sorted population (Fig. 6).
In general, we observed that α6+/MTS24+
(Fig. 6, filled bars) andα
6+/CD34+ (Fig. 6,
hatched bars) keratinocytes showed a similar gene expression profile for genes
whose expression is expected to be downregulated
(Fig. 6, red bars) and for
genes whose expression is expected to be upregulated
(Fig. 6, green bars). For
example, Dab2 (which encodes a Wnt-inhibitor)
(Hocevar et al., 2003) and
Eps8 (which encodes an EGF-pathway member)
(Miyamoto et al., 1996), whose
increased expression is associated with bulge stem cells, indeed show elevated
expression in both α6+/MTS24+ and α6+/CD34+ keratinocytes. Genes
involved in hair growth (Wnt3a)
(Millar et al., 1999) and hair
follicle differentiation (Gata3)
(Kaufman et al., 2003), whose
expression is expected to be downregulated in bulge stem cells, showed a
decreased expression in both α6+/MTS24+ and α6+/CD34+
keratinocytes. In general, we noticed that genes were more enriched withinα
6+/CD34+ compared to α6+/MTS24+ keratinocytes. One exception was
Tnc, which encodes an extracellular matrix protein, whose expression
was much higher in α6+/MTS24+ compared to α6+/CD34+ keratinocytes.
This observation was supported by immunohistochemistry (data not shown). As
expected, CD34 mRNA expression was nearly 20-fold lower inα
6+/MTS24+ keratinocytes compared to α6+/CD34+. This finding
validates our earlier observations that the MTS24 and CD34 subpopulation are
distinct cell populations within the hair follicle.

DISCUSSION

We describe a novel subpopulation of murine follicular keratinocytes that
are immunoreactive for the cell-surface marker MTS24. MTS24 is a particularly
intriguing marker based upon studies in thymic epithelium, where ectopic
transplantation of a small number of MTS24-positive thymic epithelial cells
can give rise to a complete, functional thymus, indicating that the
MTS24-positive fraction harbours epithelial stem cells
(Gill et al., 2002). Our data
show that MTS24-positive follicular keratinocytes are highly clonogenic in
vitro and have a gene expression pattern resembling that of bulge-derived
epidermal stem cells. The high proliferative capacity, stem-like expression
profile and localisation in a well-protected niche indicate that
MTS24-positive keratinocytes represent a new progenitor cell located within
murine hair follicles.

MTS24+ and CD34+ basal keratinocytes show similar gene expression
profiles. FACS-sorted α6+/MTS24+ and α6+/CD34+ keratinocytes
were analysed by Q-PCR for expression of a selected group of genes.α
6+/MTS24+ and α6+/CD34+ show the same pattern for genes that are
supposed to be lower expressed (red bars) in hair follicle stem cells or whose
expression is enriched (green bars) in hair follicle stem cells. Expression is
normalized to the reference gene (β-actin) and fold changes forα
6+/MTS24+ and α6+/CD34+ keratinocytes are in comparison toα
6+/MTS24- and α6+/CD34-keratinocytes respectively. Average data
given are from two independent isolations and Q-PCR was performed in
triplicate per sorted population.

We have shown by immunolabelling, colony-forming assays and gene expression
analysis that MTS24 and CD34 identify two distinct populations of follicular
keratinocytes that exhibit divergent in vivo characteristics and in vitro
function. The most thoroughly characterised epidermal stem cell population
resides in the bulge region of the hair follicle outer root sheath
(Cotsarelis et al., 1990;
Morris and Potten, 1994;
Lavker and Sun, 2000).
Label-retaining cells (LRCs) are concentrated in the bulge, and these cells
express the markers CD34 and keratin 15
(Trempus et al., 2003;
Morris et al., 2004). Bulge
cells are capable of giving rise to all the differentiated lineages of the
IFE, hair follicle and sebaceous gland
(Taylor et al., 2000;
Oshima et al., 2001;
Morris et al., 2004). The in
vivo observation that MTS24 cells are infrequently label-retaining when
compared to adjacent CD34-expressing cells is reminiscent of the hematopoietic
stem cell system where distinct populations of short- and long-term
progenitors have been described. As the multipotent, long-term reconstituting
hematopoietic stem cells (LT-HSC) differentiate, the self-renewal potential of
their progeny declines. Short-term hematopoietic stem cells (ST-HSC) are
multipotent and renew for 6-8 weeks; these further differentiate to give rise
to multipotent progenitors and finally oligolineage-restricted progenitor
cells (Shizuru et al., 2005).
It seems likely that a similar hierarchical restriction of lineage and
self-renewal potential may exist in progenitor cells of the epidermis. In this
context, we hypothesise that MTS24-positive keratinocytes may represent a
short-term repopulating epidermal progenitor derived from the adjacent
long-term, label-retaining CD34-expressing bulge cell population (see
Fig. 7).

Models for the relationship between the bulge and MTS24-positive hair
follicle subpopulations. The phenotype of key markers is noted for
highlighted regions. Several possibilities for the relationship between these
subpopulations are indicated in the figure. K15, keratin 15; LRC,
label-retaining cells; α6, α6-integrin; K14, keratin 14.

Despite evidence that MTS24-positive cells likely represent a short-term
repopulating subset of epidermal progenitor cells, it remains entirely
possible that these cells represent a bulge-independent population of stem or
stem-like cells that maintain a unique regenerative capacity. Several
observations generated in the course of this study support this alternative
role for MTS24-positive epidermal keratinocytes. Firstly, while MTS24-reactive
keratinocytes were already observed at E17 during hair follicle development in
wild-type mice, CD34 expression was not detected until day 6 after birth
(Fig. 2A-C). At this stage,
hair follicles are almost fully developed
(Schmidt-Ullrich and Paus,
2005). The initiation of MTS24 labelling in anagen follicles
during both the early stages of hair follicle development and
transgene-mediated ectopic hair follicle formation suggests that
MTS24-positive keratinocytes are independently involved in hair follicle
formation. Additionally, it has been suggested that LRCs located in the hair
follicle bulge are not synonymous with epidermal stem cells, but probably
represent only a subset of the total epidermal stem cell population
(Braun et al., 2003;
Claudinot et al., 2005). In
support of this, long-term lineage marking has shown that in undamaged
epidermis there are distinct stem cell populations within the interfollicular
epidermis, sebaceous glands and hair follicles
(Ghazizadeh and Taichman,
2001; Niemann and Watt,
2002). Furthermore, permanent in vivo lineage-tagging experiments
in transgenic mice have shown that bulge cells are not responsible for normal
maintenance of the interfollicular epidermis
(Ito et al., 2005;
Levy et al., 2005), and
ablation of keratin 15-positive cells results in a complete loss of the bulge
microenvironment with no effect on the interfollicular epidermis
(Ito et al., 2005). As we have
demonstrated that MTS24-positive keratinocytes are distinct from keratin
15-positive cells, it remains possible that these cells survive
transgene-mediated ablation and contribute to prolonged interfollicular
epidermal survival.

The relationship between the MTS24+ and CD34+ progenitor cell
subpopulations remains to be clarified. We propose three potential models
(Fig. 7). First, MTS24+
keratinocytes may represent a population of committed progenitor cells that
are derived from the CD34+ bulge stem cells, analogous to the
restricted-lineage progenitor populations in the hematopoietic system. The
second model suggests that the MTS24 population may represent a subset of hair
follicle stem cells that have adapted their cell-surface marker repertoire to
the local microenvironment. Interactions with the surrounding niche probably
regulate stem cell migration, proliferation and lineage specification
(Fuchs et al., 2004). The
final model is that MTS24+ cells represent a follicular stem cell population
that is completely autonomous from the CD34+ population of the follicular
bulge.

To begin to determine which of these three models best describes the MTS24+
basal keratinocytes, we compared the colony-forming efficiency in culture and
gene expression profile of MTS24+ and CD34+ basal keratinocytes. Our data show
that α6+/CD34+ bulge stem cells were approximately twice as efficient asα
6+/MTS24+ keratinocytes at forming large colonies in culture. However,
both α6+/CD34+ and α6+/MTS24+ keratinocytes showed increased
colony-forming efficiency in comparison with the unfractionated `all sorted'
population. Furthermore, both α6+/CD34+ and α6+/MTS24+
keratinocytes generated large colonies containing many small, undifferentiated
keratinocytes, and passaged efficiently to form secondary colonies of
equivalent size to each other, which provides evidence for the stem-like
nature of the two subpopulations. We analysed the self-renewal capacity of
purified keratinocytes in vitro because it has been reported that clonogenic
keratinocytes are closely related to the multipotential epidermal stem cells
(Kobayashi et al., 1993;
Rochat et al., 1994;
Oshima et al., 2001). The
results of these primary and passaged cell assays indicated an enhanced
colony-forming efficiency of MTS24-positive cells which was comparable to
established, CD34 positive bulge-associated HF stem cells. While these methods
do verify that MTS24-positive cells are epidermal progenitors, they do not
necessarily allow us to examine the differentiation potential of these cells
along sebaceous or hair-follicle lineage pathways. Future studies using ex
vivo cell engraftment will address the lineage commitment of MTS24-positive
epidermal progenitors.

Our Q-PCR results confirm that CD34 is not significantly enriched in theα
6+/MTS24+ sorted keratinocytes, providing convincing evidence that
these subsets of cells are non-overlapping. Both α6+/CD34+ andα
6+/MTS24+ keratinocytes showed a similar gene expression profile for
genes that were expected to be up- or downregulated in hair follicle stem
cells compared to non-stem cells (Morris
et al., 2004; Tumbar et al.,
2004; Claudinot et al.,
2005). However, in general, gene expression was more enriched inα
6+/CD34+ compared to α6+/MTS24+ keratinocytes. Future studies
will seek to address the effect of individual genes on the function of the
bulge and MTS24-positive cells. Taken together, the colony formation and
genetic profiling data appear to support our first model that MTS24+
keratinocytes represent a population of committed progenitor cells that are
derived from the CD34+ bulge stem cells.

The role of MTS24-positive keratinocytes in the hair follicle remains to be
analysed. In the thymus, MTS24 was reported to identify epithelial progenitor
cells that not only function to reconstitute a full epithelial compartment of
the thymus but were also able to create functional microenvironments
supporting normal T cell development
(Bennett et al., 2002;
Gill et al., 2002). We
hypothesise that the reservoir of MTS24-positive hair follicle keratinocytes
could have similar properties; i.e. MTS24-positive keratinocytes could play an
important role in organising a cellular microenvironment required for
epidermal homeostasis. Our observation that MTS24 labelling was already found
in the early stages of embryonic hair follicle development supports this
hypothesis. The location of MTS24-positive keratinocytes in a sequestered
microenvironment adjacent to the bulge and isolated from the changes that
occur in the hair follicle as it cycles, suggests that these cells are
biologically important. MTS24-positive keratinocytes appear to be
`well-placed' to produce progeny to replenish the interfollicular epidermis,
sebaceous gland and/or hair follicle lineages. To assess the lineage potential
of MTS24+ keratinocytes, it will be necessary to purify these cells and to
assess their ability to contribute to epidermal skin grafts or, preferably, to
use permanent in vivo lineage marking to assess the fate of these cells in
intact epidermis. Future work will seek to clarify the origin and role of
MTS24-positive keratinocytes during normal homeostasis and in conditions such
as skin wounding and following transplantation.

In summary, our findings demonstrate that the membrane-bound marker MTS24
selects for a novel population of follicular keratinocytes with an
undifferentiated phenotype, high proliferative potential and a gene expression
pattern resembling that of follicular stem cells. We have shown that MTS24
labelling is found in the early stages of hair follicle development and during
de novo hair follicle formation. Future experiments will seek to determine
whether the MTS24-positive keratinocytes represent a new reservoir of
epidermal stem cells or a population of lineage-restricted progenitor cells.
Either outcome would be interesting since markers for progenitor keratinocytes
have yet to be identified within the hair follicle. Furthermore,
characterisation of the molecular and functional attributes of MTS24-positive
epidermal cells may provide targets for modifying keratinocyte progenitor cell
behaviour in circumstances such as alopecia, wound healing and cancer.
Finally, MTS24 has now been reported as a putative marker of both thymic and
epidermal progenitor cells. Therefore, elucidation of the functional
properties of the MTS24 cell-surface antigen will probably provide broad
insights regarding progenitor cell biology of multiple epithelial organs.

Supplementary material

Acknowledgments

This work was supported in part by a grant from the Dutch Cancer Society
(J.N, RUL 2002-2737), EuroStemCell (K.B.) and Cancer Research UK (F.M.W., A.G.
and K.B.). A.G. is the recipient of a Marshall Sherfield Fellowship and NIH
fellowship. We thank Jos Onderwater and Aat Mulder from the LUMC Center of
Electron Microscopy for performing the immuno-electronmicroscopy work; Wim
Zoutman from the Department of Dermatology for assisting with the Q-PCR gene
expression profiling and Kyoko Masuda from the RIKEN Laboratoryfor Lymphocyte
Development for initial cell-sorting experiments. We are grateful to the
Cancer Research UK Biological Resources, Histopathology and FACS Units (in
particular Kirsty Allen) for expert technical assistance.